مجلة إبن الھیثم للعلوم الصرفة و التطبیقیة 2012 السنة 25 لدالمج 1 العدد Ibn A l-Haitham Journal f or Pure and Applied Science No. 1 Vol. 25 Year 2012 Turbidimetric Method for Measuring o f Antibacterial Effect of 2-Methyl-4-(3,4 dihydroxy Phenyl)-Thiazole on the Growth Activity of Escherichia Co li and Pseudo mo nas Aerugino sa A.K. Abbas Departme nt of Physiological Chemistry, College of Medicine. Unive rsity of Baghdad Re ceived in : 26 April 2009 Accepted in : 18 August 2009 Abstract Thiazoles are heterocyclic nitrogenous comp ounds, which were given a gr eat att ention due to their antibacterial effect, antihyp ertensive, stimulating glucose absorp tion (like antibiotics), acts as cytop rotective agent. T he present work is conducted to evaluate the degree of antibacterial activity on the growt h activity of Es ch er ich ia C o li ( E. C o l i) and Ps eu d o m o n a s a er u g in o s a ( P. a er u g in o s a ) b y incub ation with 2-methy l-4-(3,4 dihydroxy p henyl)-Thiazole comp ound (M DHPT). Opt ical density (O.B) was measured by turbidmetric method, any increase in O.D would represent the increase in the bacterial growt h .The results showed that there was a 75.7 % inhibition when usin g 190.4 g/ml (M DHPT) solution and 64.3% was obtained when usin g concentration of 369 g/ml. It has been concluded that (MDHPT ) comp ound shows a bacterial b iolo gical activity . Key words: Thiazoles, E . C o l i, P . aer u gin o s a, i n c u b a t i o n m e d i u m , incubator, minimu m inhibitory concentration (M IC) Introduction The st udy of new derivative of Thiazole has been given a great att ention, due to their antibacterial activity [1, 2, 3] antihypertensive [4] and st imulation of glucose absorp tion like antibiotics [5], lowerin g cholest erol [2], shows bacteria st atic activity [3] and acts as cytop rotective agent by induction of glutathione-S-transferases (GST) represent not only cell detoxification and survival but also cancer p revention [6,7]. Sever al att emp ts were carried out to elucid ate the effect of different antibiotics relate d to this comp ound on the growt h activity of p athogenic St ap hy lo co ccu s au r eu s [2,3,8,9,10,11,12,13,14] This work is related to the action of 2-M ethy l-4-(3,4 dihydroxyp henyl)-thiazole (MDHPT) on t he growt h activity of Es ch er ich i a C o li ( E. C o l i) and Ps eu d o mo n a s a e ru g i n o sa ( P. a e ru g i n o sa ) either in solid or in liquid media . مجلة إبن الھیثم للعلوم الصرفة و التطبیقیة 2012 السنة 25 لدالمج 1 العدد Ibn A l-Haitham Journal f or Pure and Applied Science No. 1 Vol. 25 Year 2012 Both bacteria are of normal flora of the intestine and they cause different diseases esp ecially in weak p eople [15]. The clinical records revealed that 83% of p atients with leukemia havin g infection with P s eu d o mo n as aer u g in o s a [16].This bacteria is also resistant to many antibiotics which may be attributed to alteration in cell membrane transp ort sy stem. [17]. The aim of this st udy is to evaluate the degree of the antibacterial inhib itory effect of 2- M ethy l-4-(3,4 dihydroxyp heny l)-Thiazole on the growt h activity of E. C o li and P . aer u g in o s a and to find the effective dose r equired . M ate ria ls and M e thods 2-M ethy l-4-(3,4 dihydroxyp heny l)-Thiazole hy drochloride, was sy nthesized by p rofessor Shukri, J, Dep artment of chemistry , College of Scien ce , University of Baghdad [1]. (2-M ethy f-4-(3,4 dihydroxyp heny l)-Thiazole) A stock solution was p rep ared by dissolving (4 x 10 3 g/ml) in water with few drop s of glycerol. To p repare a starting culture of bacteria, a loop full of st ock culture from each typ e of bacteria was added to 50 ml of try ptose p hosp hate broth medium (29.5 g/liter, p H 7.3) and incubated in shak ing water bath for 20-24 hours at 37° C and at 200-250 rp m (rotation / minute ). Exp erimental flasks of 250 ml with side arm, each contains 20 ml of st erilized cultur e medium. Each flask was ino culated with 1.0 % ( V/V) of both used bacteria susp ension which contain 2.4 x 10 7 cell / ml [18]. The number of bacterial cells was calculated by using Neub er heamocy tometer [19]. (M DHPT) solution was added to the inoculated media at the beginning of cultivation, the flasks were closed with cotton p lugs under st erilized conditions. A control was run under the same conditions without the addition of thiazole solution. The flasks were p ut in the shaking incub ator at 200-250 rp m to ensure adequate aeration at 37 o C for 24 hours. Changes in the concentration of v iable bacteria dur ing lag, exp onential and st ationary p hases were measured by chan ges in the op tical density at 607 nm, usin g EEL colorimeter (Evans Electroselenium LTD. Halstead, Essex, England). مجلة إبن الھیثم للعلوم الصرفة و التطبیقیة 2012 السنة 25 لدالمج 1 العدد Ibn A l-Haitham Journal f or Pure and Applied Science No. 1 Vol. 25 Year 2012 R es ults Different concentrations of 2-M ethy l-4- (3,4- dihydroxy p henyl)-thiazole ranging from (19.9 g/ml (8.1 x 10 -2 ) - 190.4 g/ml (7.8 x l0 -1 M )) were added to E. C o li culture media (after six hours of incubation). The results showed an inhibition of (24.3, 37.8, 48.6 and 75.7 %) in the growt h rate of the bacterial culture, at concentrations of (19.9 g/ml (8.1 -2 M ), 59.1 g/ml (2.4 x 10 -1 M ), 97.5 g/ml (4 x 10 -1 M ), and 190.4 g/ml (7.8 x 10 -1 M )) resp ectively; Table 1, Fig. l. Inoculation with P. a er u g in o s a required higher (M DHPT) concentrations than that used with E. C o li at the same conditions. Concentrations of (135.2 g/ml (5.5 x 10 -1 M ), 244.4 g/ml (l.0 M ), 313.4 g/ml (1.28 M ) and 369.6 g/ml (1.49 M )) showed an inhibitory effect of (16.13, 38.7, 48.39, and 64.3%) r esp ectively in comp arison to control activity for E. C o li and P. a er u g in o s a . At the same exp erimental conditions Table 2, Fi g 2. The results were the mean of ten exp eriments with P< 0.05. All data are exp ressed as a mean of ten exp eriments with P< 0.05. D is cus s ion (M DHPT ) was st udied with St ap h y lo co cu s au r eu s results show bacteria st atic activity of 70% inhibition (M IC 200 g/ml = 0.82 M ) [2,3]. In this st udy a bacteria st atic activity of 75.7 % was achieved with a concentration of, 190.4 g/ml (8.1uM ) inoculated, with E. C o li in liquid medium after 6 hours of incubation time. This results were inconsistent with what was reported by (Faraid, Al-Chalabi et al, and Shukri, J. et al), while a higher concentration solution of (M DHPT ) 313.4 g/ml (l28 M ), was required to achieve nearly the same rate of inhibition (64.3 %) as with E. Coli [2, 3 ]. Bacteria st atic activity was achiev ed, this may be exp lained by the p resence of p otent group (s) t hat achieved the inh ibitory action on the growt h activities of each of gram- negative E. C o li and P. a er u g in o s a , and also gr am-p ositive S ta p h ylo co ccu s a u r eu s ( S t. a u r eu s ) . The p resence of active M ethy l group (-CH3) at p osition 2 of the Thiazole ring which can react with carbonyl group s of t he peptidoglycan or lipop oly saccharide (LPS) of the bacterial cell membrane [2, 20, 21]. The dissociation of p henolic group s p resent in the comp ound (M DHPT) may cause a release of excess p rotons (H + ) to the medium which may be p icked up by the (-NH2) of the p ep tidoglycan of the bacterial cell membrane formin g excess (-NH + 3) which may create an ionic attraction lead ing to a cell wall d amage[3,19]. The cell wall of gram-negative bacteria has d istinctly lay ered appearance, the inner region consists of monolay er of peptidoglycan, while the outer cell wall b ilayer inward facin g lip ids are macromolecules called lip op olysaccharides ( LPS), h alf the mass of the outer membran e consist s of Braun p roteins which covalently linked to p eptidoglycan lay er, adjacent outer lip op olysaccharides are h eld together by electrost atic interaction with divalent ions ( Zn +2 , Ca +2 , M g +2 ), while gr am-p ositive cell wall is multi-lay ered network which ap p ears t o be continually-growin g by the addition of new p eptidoglycan linear hetrop oly saccharide chain cross-linked by short p olyp ep tides at the interface with concomitant out er surface .[21,22,23]. There may be another p ossibility to exp lain the inhibitory effect it-may-be that the activity of (M DHPT) is due to its ability to chelate essential metal ions e.g. (Zn, M g +2 ) from bacteria whi ch cause a d isturb of these interaction of gr am-negative ( E. C o li & P. a er u g en o s a ) by binding to the divalent ions in the cell wall which . cause the disintegration. Also chelation may occur to those ions that required for enzymes activation مجلة إبن الھیثم للعلوم الصرفة و التطبیقیة 2012 السنة 25 لدالمج 1 العدد Ibn A l-Haitham Journal f or Pure and Applied Science No. 1 Vol. 25 Year 2012 p rocesses occurred in bacterial cell wall. [ 20,21,22]. Variation in the results obtained with both bacteria under st udy could be due to high variation in amounts of the p eptidoglycan p resent in the gram-n egative or gram positive bacteria cell wall membrane [21,24]. Re ferences 1. Shukri, J. (1977) Brenzcatechin substituierte, Thiazofe, Wiss, z. Univ. Halle, xxVI, 77M ,H,5(S): 59-61. 2. Al-Chalabi, F.A.J.; Elham, M .H.; Jaafar and Az iz, HH, Al-Lami, (1984) Effect of new thiazole derivatives on the growt h of st aphy lococcus aureus, J.B.C. Vol. 15 (2). 3. Shukri, J, Shubber, A; Al-Chalabi, FAJ, Khalif a, A and Hassan, F. (1979) New thiazole comp ounds as p otential antibacterial agents. Iraqi J. Sci., 20 (l):75-86. 4. Smith-Klein and French Laboratories, (1972) Phil. USA, Private report. 5. Al-Lami, AHH; Al-Chalabi, FAJ, and Jaafar, M H, (1983) The influence of new thiazole comp ounds on glucose absorp tion from isolated intest inal segments of the rabbits. J. Biol. Sc. 214(1): 3-14. 6. Sang Geon Ki m and Seun g Jin Lee, (2007) Toxicolo gical Sciences, 96 (2) 206-213 ; doi: 10, 1093 Tox Sci/K 1175. 7. Kim, H.P. Ryt er, S.W., and Chei, A,M ., 2005; Annu.Rev. Pharmacol. To xico l. 8. Collins, J.F. (1974) App lication of Prokary otic Genetics in Biochemical Studies, in Companion to Biochemistry Bull E, Lagnado JR, Thomas JO, and Tripton KF; pp .471, Longman, London. 9. Eagle, H. and M isselman, A.D.(1948) The rate of bactericidal action of p encillin in vitro as a function of its concentration and its p aradoxically reduced activity at high concentrations against certain or ganisms.J. Exp tl. M ed. 88:99-131. 10. M ay hall, M . and Ap ollo, E. (1980) Effect of storage and chan ges in bacterial growt h p hase and antibiotics concentrations on antimicrobial toler ance in st aphy lococcus aureus,Antimicrobial-agents chemother., 18(5):784-788. 11. Peterson, E.; Fairahter, M orrison, J. and Cesario, T. (1981) Effect of the herbicide p araquate dichloride on bacterial of human origin. Ap p l. Environ. M icrobial: 41(l): 327-328. 12. Raynor, RH; Scott , DF, and Best, Co.K. (1976) Oxicillin-Induced lysis of st ap hy lococcus aureus.Ant imicrobial. A gents Chemother. 16:134-140. 13. Watnakunakron, C. and Cuerriero, J.C.(1981) Interaction between vanco mycin and rifampin against st aphy lococcus aureus. Ant imicrobial. A gents Chemother.: 19(6) : 1089-1091. 14. Jawad, A.M . and Jaffer, H.J. (2008) In vitro antimicrobial activity of total sequiter p enlactones and phenols isolated from some Iraqi plants. Um salama. Scien ce Journa l, 5(1): 80-83. 15. Ernest Jawetz, M elnick, JL.Adelberg, EA (1974) Review of medical microbiology , 14 th Ed., Lange medical publication Canada; p . 211-212. 16. M icheal, V. Viola(1967)Acute Leukemia and Infection. J.A.M ., 201-203. 17. Sp icar, A.B.(1976) Increased antibiotic sensitivity in p seudomonas aeru ginosa followin g p assage in carbenicillin containin g media. J. App . M icrobial., 40: 33-45. 18. Jaafar, E.M .H.(1976) A st udy on drug resistance Saccaromyces Cervisiae M .Sc. Thesis, Warwick University England. 19. Cruickshank, R, Dugu id, JP, M armion, BLA, M edical M icrobiolo gy , Vol. 2, 12 th Ed. مجلة إبن الھیثم للعلوم الصرفة و التطبیقیة 2012 السنة 25 لدالمج 1 العدد Ibn A l-Haitham Journal f or Pure and Applied Science No. 1 Vol. 25 Year 2012 20. Pamela,C. Ch amp e and Richard A Harvey "Lipp incott's Illust rated Reviews: Biochemist ry , 2 nd ed. Copy right (1994) J.B. Lippincott Comp any . 21. M ichaels Blader, Sp ring. (1998) BCH 5425 M olecular Biolo gy and Biotechnolo gy 16:925-928. 22. Al-Chalabi, F.A.; Al-Najjar, F.A.; Al-Ani, S.M. The effect of some metal ions and their chelates on the viability of staphy lococcus aureus J Ibn Al-Haitham, M Pure and App Sci., Vol. 5(1): 23. Al-Chalabi, F.A.; M ehdi, W.K. and Al-Karaghouli, K.(2003) The effect of Nickel celates on the reactivation of inh ibited Staphy lococcus. 24. Huang, J.W. ; Blaylock, M .J.; Kap ulnik, X and Ensley , B.D. (1998) Environ., Sci. Technol. 32:2004-2008. Table (1): Represents counts of viable E. C o li cells and % inhibiti on when 2-Methyl-4- (3,4 dihydroxyphe nyl ) -Thiazole compound was added to inocul ation me dium compared to control. Mean of ten e xperiments P< 0.05 Time hours Test No. of viable E. C o l i Cells Added 1.99 g/ml (0.8 M) (MDHPT) % Inhibition Control 0 2.4 x 10 7 0.0 2.4 x 10 7 2 2.6 x 10 7 2 4 . 3 3.6 x 10 7 4 7.2 x 10 8 32.8 3.6 x 10 9 6 3.5 x 10 9 48.6 8.8 x 10 9 24 4.9 x 10 12 75.7 7.0 x 10 12 مجلة إبن الھیثم للعلوم الصرفة و التطبیقیة 2012 السنة 25 لدالمج 1 العدد Ibn A l-Haitham Journal f or Pure and Applied Science No. 1 Vol. 25 Year 2012 Table (2): S hows counts of viable P. aeruginosa cells and % inhibition when 2-Methyl -4- (3,4 dihydroxyphe nyl )-Thiazole compound was added to inocul ation me dium, compared to control. Mean of ten e xperiments P< 0.05 Time hours Test No. of viable P. aeruginosa Cells Added 369.6 g/ml (1.49 M) (MDHPT) % Inhibition Control 0 2.4 x 10 7 0.0 2.4 x 10 7 2 1.5 x 10 8 16.13 8.6 x 10 7 4 1.3 x 10 9 38.7 8,0 x 10 9 6 3.9x 10 10 48.39 8.0 x 10 10 24 5.8x 10 11 64.3 9.0x 10 11 Fig. (1): Effect of various concentrations of 2-Methyl-4 3,4 dihydroxyphe nyl ) 4- Thi azole hydrochloride on the growth activity of E. Col i a- Control bacterial growth of E.Coli, b-Inocul ation medium contains 8.1 x 10 -2 uM of (MDHPT). c- contains 2.4 x 10 -1 M (MDHPT), d- (MDHPT) 4 x 10 -1 M e- (MDHPT) 7.8 x x 10 -1 . Ex perime ntal conditions were described in the method. Data are expressed as a mean of ten experiments , P<0.05, مجلة إبن الھیثم للعلوم الصرفة و التطبیقیة 2012 السنة 25 لدالمج 1 العدد Ibn A l-Haitham Journal f or Pure and Applied Science No. 1 Vol. 25 Year 2012 Fig. (2):Effect of va rious concentrations of 2-Methyl-4- (3,4 dihydroxyphe nyl ) - Thiazole hydrochl oride (MDHPT) on the growth activity of P. aeruginosa a-Control bacterial growth, b- (MDHPT) 5.5 x 10 - 1  M. c- (MDHPT) 1.0 M. d- (MDHPT) 1.2 M e- (MDHPT) 1.49 M. Ex perime ntal conditions as in F i g l . P< 0.05. مجلة إبن الھیثم للعلوم الصرفة و التطبیقیة 2012 السنة 25 لدالمج 1 العدد Ibn A l-Haitham Journal f or Pure and Applied Science No. 1 Vol. 25 Year 2012 مستوى النمو للبكتریا فيتأثیر مضادات البكتریا لمركبات الثایوزول )Pseudomonas aeruginosa و Escherichia co li( س احسان خلیل عبا قسم الكیمیاء ، كلیة الطب ،جامعة بغداد 2009 آب 18: قبل البحث في2009نیسان 26: استلم البحث في الخالصة ت تعد مركبات الثایوزول العضویة من المركبات التي أعطیت أهمیة كبیرة في ضوء خصائصها بوصفها مضادا ع ًبكتیریة وحیویة فضال عن كونها محضرات لعملیة امتصاص سكر الكلوكوز وتقدیر فعالیة التثبیط لنمو البكتریا لألنوا )Pseudomonas aeruginosa و Escherichia coli ( باستعمال مركبات الثایوزول العضویة حاضنات فقد استعمل .لهذا الغرض) MDHPT( ثایوزول –) ثنائي هیدروكسي فنیل3,4 (– 4 –المركب العضوي ثنائي المثیل . قیاس الكثافة الضوئیة تعني ازدیاد تثبیط نمو البكتریا التي تم اختیارها في هذه الدراسةأظهرت النتائج من خالل بینما كانت النسبة %) 75.5(لها فعالیة تثبیط بنسبة ) g/ml 190.4(كما أثبتت النتائج بأن تركیز مادة الثایوزول ًان له تأثیرا كبیرا ) MDHPT(تي عد المركب وفي ضوء النتائج ال) g/ml 369(عند التركیز %) 64.3(المثبطة للنمو ً .في نمو البكتریا M(، حاضن ، أقل تركیز تثبیطي )حاضن(مركب الثیازول، وسط حراري : الكلمات المفتاحیة IC( مجلة إبن الھیثم للعلوم الصرفة و التطبیقیة 2012 السنة 25 لدالمج 1 العدد Ibn A l-Haitham Journal f or Pure and Applied Science No. 1 Vol. 25 Year 2012